Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum–infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.

Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence.

The soilborne pathogen Ralstonia solanacearum (Rs) causes a lethal bacterial wilt disease of tomato and many other crops by infecting host roots, then colonizing the water-transporting xylem vessels. Tomato xylem sap is nutritionally limiting but it does contain some carbon sources including sucrose, trehalose, and myo-inositol. Transcriptomic analyses revealed that Rs expresses distinct catabolic pathways at low cell density (LCD) and high cell density (HCD). To investigate the links between bacterial catabolism, infection stage, and virulence, we measured in planta fitness of bacterial mutants lacking specific carbon catabolic pathways expressed at either LCD or HCD. We hypothesized that early in disease, during root infection, the bacterium depends on carbon sources catabolized at LCD, while HCD carbon sources are only required later in disease during stem colonization. An Rs ΔiolG mutant unable to use the LCD-catabolized nutrient myo-inositol was defective in tomato root colonization, but after it reached the stem this strain colonized and caused symptoms as well as wild type. In contrast, Rs mutants unable to use the HCD-catabolized nutrients sucrose (ΔscrA), trehalose (ΔtreA), or both (∆scrA/treA) infected roots as well as wild type Rs but were defective in colonization and competitive fitness in mid-stems and had reduced virulence. Further, xylem sap from tomato plants colonized by ΔscrA, ΔtreA, or ΔscrA/treA Rs mutants contained twice as much sucrose as sap from plants colonized by wild-type Rs. Together, these findings suggest that quorum sensing specifically adapts Rs metabolism for success in the different nutritional environments of plant roots and xylem sap.

The soilborne pathogen Ralstonia solanacearum (Rs) causes lethal bacterial wilt disease of tomato and many other crops by infecting host roots and then colonizing the xylem vessels. Tomato xylem sap is nutritionally limiting but it does contain sucrose and trehalose. Transcriptomic analyses revealed that Rs expresses distinct sets of catabolic pathways at low cell density (LCD) and high cell density (HCD). To investigate the links between bacterial catabolism, infection stage, and virulence, we measured the in planta fitness of bacterial mutants lacking carbon catabolic pathways expressed at either LCD or HCD. We hypothesized that the bacterium needs LCD carbon sources early in disease (root infection) while HCD carbon sources are required during late disease (stem colonization). An Rs ΔiolG mutant unable to use the LCD nutrient myo-inositol was defective in root colonization but once it reached the stem, this strain colonized and caused symptoms as well as wild type. In contrast, Rs mutants unable to use sucrose (ΔscrA), trehalose (ΔtreA), or both (∆scrA/treA), infected roots as well as wild type but were defective in colonization and competitive fitness in tomato mid-stems and were reduced in bacterial wilt virulence. Additionally, xylem sap from tomato plants colonized by ΔscrA, ΔtreA, or ΔscrA/treA contained more sucrose than sap from plants colonized by wild-type Rs. Together, these findings suggest Rs metabolism is specifically adapted for success in the different nutritional environments of plant roots and xylem sap.